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A.F. Clark, C. Millar, I.–H. Pang, N. Jacobson, A.R. Shepard; Adenoviral Gene Transfer of Active Human Transforming Growth Factor–ß2 Induces Elevated Intraocular Pressure in Rats . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4771.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor–ß2 (TGFß2) has been implicated in the pathogenesis of glaucoma based upon its elevated levels in glaucomatous vs. normal aqueous humor and its known role in inducing extracellular matrix remodeling proteins. Our goal was to generate a rodent model of primary open–angle glaucoma using adenoviral gene transfer of human TGFß2.
Latent (wild–type) or active (C226S, C228S) TGFß2–encoding cDNA (RefSeq NM_003238) was cloned into the pac.Ad5.CMV.K–N.pA shuttle vector for generation of replication–deficient adenovirus. Empty adenovirus (Ad5.CMV.K–N.pA) was used as a control. Adenoviral expression of active and total (acid–activated) TGFß2 was initially checked in vitro by transduction of GTM3 cells and measurement of TGFß2 levels in the cell media by ELISA. Wistar rats injected intracamerally with the adenovectors were assessed for changes in intraocular pressure (IOP) using the TonoLab rebound tonometer. At various end–points, active and total TGFß2 levels in aqueous humor were measured by ELISA. Expression of the exogenous adenoviral TGFß2 mRNA in the Ad5.CMV.K–N.pA.TGFß2 injected eyes was also confirmed by RNA isolation from anterior segment tissue and qRT–PCR analysis.
We first overexpressed the latent form of human TGFß2 by intracameral injection of the adenovirus into rats. IOP was not significantly elevated (p>0.05) with the latent form, which was likely due to the low in situ activation of the molecule. We next overexpressed TGFß2 containing two mutations in the latency associated peptide portion of the molecule, resulting in production of spontaneously active protein and significantly elevated IOP (p<0.05) from a baseline of 18.0 ± 0.4 mmHg (mean & SEM, n = 10) to 25.4 ± 2.4 mmHg in the rodent.
We have generated a spontaneously active form of human TGFß2 that, when expressed by adenoviral gene transfer into rat eyes, causes significantly elevated IOP.
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